Possible cytotoxic mechanisms of bis-(2-chloroethyl)sulfide (BCES) in rat keratinocytes in relation to DNA alkylation.

Abstract

Two hypothetical mechanisms for the cytotoxicity of bis-(2-chloroethyl)sulfide (BCES) were evaluated in cultures of rat keratinocytes. The first hypothesis was that repair of BCES-mediated DNA-breaks involving poly(ADP-ribose)polymerase, which uses NAD$\sp+$ as a substrate, depletes the level of NAD$\sp+$ resulting in cell death. However, although the content of DNA was reduced 48 hr after exposure to 10 $\mu$M BCES in both proliferating and early differentiating cultures, the total level of NAD$\sp+$ plus NADH was not changed. Furthermore, supplementation of medium with 1-10 mM nicotinamide, an inhibitor of poly(ADP-ribose)polymerase, increased the level of NAD$\sp+$ plus NADH of cultures exposed to 250 $\mu$M BCES from approximately 24% to 40-45% of control at both 24 and 48 hr post-exposure, but did not reverse the decrease in cellular DNA from 70% to 35% of control. Thus, no evidence in support of the "NAD depletion" hypothesis was obtained. The second hypothesis, that interstrand cross-links in DNA prevent the replication and repair of DNA ultimately leading to cell death, was evaluated by analyzing the cell cycle in relation to cross-linking of DNA. When keratinocytes synchronized by aphidicolin were exposed to BCES in different phases of the cell cycle, cells in S phase appeared to be more sensitive to BCES than cells in other phases. Keratinocytes synchronized at the G1/S border and exposed to 1 $\mu$M BCES exhibited a prolongation of the S phase and a block in the G2 phase. When exposed to 10 or 50 $\mu$M BCES, cultures were inhibited from entering S phase and the incorporation of thymidine into DNA was also inhibited. Immediately after exposure, 1-100 $\mu$M BCES induced a dose-dependent formation of DNA interstrand cross-links. At 24 hr post-exposure, cross-linkage in cells at the G1/S border was slightly reduced. However, cross-links in G1 phase-exposed cells was dramatically reduced. The antiproliferative effect of BCES could be a consequence of blocks in the G1 and G2 phases, and the interstrand cross-links in DNA may be responsible for this effect.